Vitamin K is an essential nutrient in the diet of humans. Its role in the posttranslation carboxylation of blood-coagulation proteins and other proteins involved in the mineralization of bone has only recently been elucidated. The enzyme, Vit.K dependent carboxylase, catalyzes the formation of gamma carboxyglutamic acid which is a specific calcium binding site on the coagulation and bone proteins. The K dependent carboxylation reaction is complex. Substrates include reduced Vit. K1, O2, CO2, and either precursor proteins or synthetic glutamic acid containing proteins. Research will include studies on the stoichiometry and mechanism of this reaction with the ultimate goal of understanding the catalytic significance of potential intermediates such as Vit. K. hydrooperoxide and Vit. K-Glu peptide adducts. To achieve these goals we are developing a new assay system for K carboxylase which involves the use of high pressure liquid chromatography. Other techniques which will be used to elucidate the mechanism of K carboxylase catalysis will include stable isotope (18O and 13C) exchange, kinetics, and the use of chemical model systems which mimic parts of the more complex enzymatic reaction. In addition, we propose to see if gamma carboxyglutamic acid exists in a Ca ions regulatory protein responsively, these studies are important to a better understanding of human nutrition and health. Vitamin K and the enzyme with which it functions serves an important regulatory function as they control the post-translational modification of the physiologically important blood-clotting and bone mineralization proteins.